RESUMO
Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Lactobacillus/fisiologia , Polifosfatos/metabolismo , Estresse Fisiológico , Meios de Cultura/química , Deleção de Genes , Concentração de Íons de Hidrogênio , Lactobacillus/enzimologia , Lactobacillus/genética , Lactobacillus/metabolismo , Microscopia , Pressão Osmótica , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , SaisRESUMO
Lactobacillus casei can metabolize L-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization of L-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on L-malic acid. In this work, we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis. Our results show that ME is essential for L-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on L-malic acid, thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of L-malic acid and repressed by glucose, whereas TC system expression was induced by L-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats [5'-TTATT(A/T)AA-3'] in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.
Assuntos
Proteínas de Bactérias/fisiologia , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/metabolismo , Malatos/metabolismo , Proteínas Quinases/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Proteínas de Bactérias/genética , Pegada de DNA , DNA Bacteriano/metabolismo , Enterococcus faecalis/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Histidina Quinase , Redes e Vias Metabólicas , Família Multigênica , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Quinases/genética , Homologia de Sequência de Aminoácidos , Streptococcus/genética , Fatores de Transcrição/genéticaRESUMO
Recently, the industrial and academic interest in the yeast Torulaspora delbrueckii has increased notably due to its high resistance to several stresses. This characteristic has made of this organism a very attractive model to study the molecular basis of the stress response in yeast. However, very little is known about the physiology and genetics of this yeast, and the tools for its manipulation have not been developed. Here, we have generated Ura(-) strains of the baker's yeast T. delbrueckii IGC5323 by either 5-FOA-aided selection or transformation with a PCR-based disruption cassette, natMX4, which confers nourseothricin resistance. Furthermore, the mutant and disruptant strains were used as recipient of a plasmid containing the xlnB cDNA from Aspergillus nidulans. Our results indicate that Torulaspora transformants produce active recombinant protein at a similar level to that found for Saccharomyces.
